Garg, Swati (2019) Evaluation of Production of Opioid Peptides from Wheat Proteins. PhD thesis, Victoria University.

Abstract

Opioids such as morphine and codeine are the most commonly clinically used drugs for pain management, but have associated side-effects. Food-derived opioid peptides can be suitable alternative due to less side-effect and are relatively inexpensive to produce. So, wheat protein (gluten) was tested as source for production of opioid peptides. The thesis reports results of investigations carried out on production of opioid peptides from wheat gluten using enzymatic hydrolysis and fermentation by selected lactic acid bacteria and characterisation of bioactivity (opioid) of prepared peptides and gluten hydrolysates. Gluten protein sequences were accessed using in silico approach (Biopep database and PeptideRanker) to predict presence of opioid peptides. The search was based on presence of tyrosine and proline. This led to selection of three peptides for which opioid activity was measured by cAMP (cyclic adenosine monophosphate) assay. The EC50 values of YPG, YYPG and YIPP were 1.78 mg/mL, 0.74 mg/mL and 1.42 mg/mL for μ- opioid receptor, respectively. Hydrolysates from gluten were produced using two different approaches, fermentation using lactic acid bacteria (LAB) and by commercial proteases. Six LAB (Lb. acidophilus, Lb. alimentarius, Lb. brevis, Lb. fermentum, Lb. plantarum and Lb. hilgardii) were selected based on their proteolytic activity and fermentation was carried out at 37°C for 24 h. After fermentation, hydrolysis was assessed by ophthalaldehyde (OPA) assay and gel electrophoresis. Reverse phase HPLC and Size exclusion HPLC were used for peptide profiling. Lb. acidophilus, Lb. brevis, Lb. fermentum and Lb. plantarum showed more proteolysis. EC50 values of <3 kDa hydrolysate fractions after fermentation were 6.3, 7.2, 4.9 and 4.3 mg/mL, respectively. Effect of selected enzymes (pepsin, trypsin, alcalase and flavourzyme) on production of opioid peptides was assessed through similar approach. The hydrolysates fraction produced by flavourzyme was the best with EC50 value of 0.43 mg/mL. The number of peptides showing > 0.5 ranking were 19, 48 and 61 for pepsin, alcalase and flavourzyme fractions, respectively. Peptides, PQQPFPL from pepsin hydrolysate and QQPPFW and QPFPQPQPFP from flavourzyme hydrolysate showed ranking ≥ 0.9 and should be further tested for bioactivity. Difficulty in dispersion of gluten due to clumping was observed during experiments involving enzymatic hydrolysis. This was investigated by characterising gluten dispersion as a function of acidic pH (1, 2 and 3) and hydrothermal treatment in terms of structural changes by scanning electron microscopy, fourier transform infra-red (FTIR) spectroscopy, changes in functional groups (free amino and thiol), and size distribution (sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and SDS extractability). Decrease in the extractability and free thiol and free amino group at pH-2 and 3 confirmed gluten complex formation. Also, hydrothermal treatment at pH-1 can be used as pre-treatment before enzymatic hydrolysis to improve peptide yield. The study concludes that wheat gluten can be used for the production of opioid peptides.

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